Any discussion of the prior art throughout the specification should not be considered an admission that such prior art is widely known or forms part of common general knowledge in the field.
Stable maintenance of molecules, and in particular functional molecules, on support surfaces can be critical for analytical and diagnostic tools—for example, in high throughput analysis and functional characterization of biomolecules. However, techniques for stably immobilizing and preserving functional proteins, biomolecules and biosensors on support surfaces have not been optimised.
For DNA chips, designing capture molecules and developing read-out systems is relatively straightforward. DNA or oligonucleotides bind specifically to complementary mRNA sequence tagged with a fluorescent dye. However, designing arrays for proteins is more complex and requires different consideration. Proteins must be immobilized on the surface of the chip such that they retain their native conformation and also such that their active site(s) are exposed rather than buried. Constructing a protein chip therefore involves more steps and more complex protein chemistry. The following are some of the issues to consider when building a useful protein chip: 1) immobilization of proteins of diverse types such that they retain their secondary and tertiary structure, and thus their biological activity; 2) identification and isolation of an agent with which to capture the proteins of interest; 3) a means of measuring the degree of protein binding, ensuring both sensitivity and a suitable range of operation; and 4) extraction of the detected protein from the chip and analysis of the protein.
Many prophylactic and therapeutic agents require special conditions to protect them from environmental damage during transportation and storage. This adds to the cost of the drug and, in certain instances, reduced availability in some communities eg. in remote and/or disadvantaged communities where conditions such as low temperature cannot easily be achieved during transportation over long distances.
Further, there is a continuing need for new pharmaceutical formulations, excipients, and delivery devices, including nanodevices, to achieve maximum benefit from drugs. For example, slow release and/or organ-/tissue-specific release of prophylactic and therapeutic agents can provide increased drug efficacy, improve bioavailability and reduce drug dosage. There is a need for versatile and safe means for administering agents intact and with site-specific and dose-specific accuracy.
Cytoplasmic Polyhedrosis Viruses
Insect virus infections result in the production of massive amounts of large protein crystals (occlusion bodies), termed polyhedra and many virus particles are occluded within the polyhedra. An insect virus, cypovirus (cytoplasmic polyhedrosis viruses, CPV), is classified among the family Reoviridae and has a segmented genome composed of ten double-stranded RNAs. The virion consists of an icosahedral protein shell of 50-70 nm in diameter and twelve spikes on the 12 vertices of the shell. Polyhedra are the main vectors of virus particle transmission from insect to insect and are the main agents of survival of the virus between one insect generation and the next because they stabilize virions, allowing them to remain viable for long periods in the environment. Infection occurs when an insect ingests the alkali-soluble occlusion body and the virus particles are released by the high pH of the insect intestine.
The virus particles of CPVs and baculoviruses produce micro-sized protein crystals called polyhedra in which virus particles can be embedded (Belloncik and Mori 1998; Miller 1997). Polyhedra exhibit remarkable stability and, as such, the embedded insect viruses can remain infectious for years in the environment. The virus particles are embedded in the polyhedron via the constituent CPV envelope protein, VP3, which binds to the polyhedrin protein, the major viral protein making up the polyhedron.
The improved stability of viral particles within the polyhedra has prompted studies investigating whether target proteins can be embedded within polyhedra for improving their stability and facilitating their use in protein microarray applications (US20060155114 and Ikeda et al 2006). This approach involved co-expressing target proteins fused to a portion of a virion structural capsid protein, VP3 during CPV infection. The results indicated that incorporation of these proteins into the polyhedron crystal was successful and that the proteins were protected from dehydration and stabilized against high temperatures without the loss of function. Proteins that have been incorporated into polyhedra using the VP3, are non-membrane and membrane proteins including, enzymes such as polymerases, kinases and acryltransferases; structural proteins such as ribosome proteins and ribosome binding proteins; and transcription factors; and elongation factors. Accordingly, the skilled addressee would understand that a diverse range of different types of molecules may be incorporated into a polyhedron without the loss of function.
Although these unique protein crystals have been characterised since the early 1900s (Glaser and Chapman 1916) determination of their atomic structure which would allow for the further development of the polyhedra into useful diagnostic, therapeutic and research tools has been elusive. Further there has been no suggestion that exogenous molecules such as proteins can be incorporated into a polyhedron crystal in the absence of a virion structural protein tag. There has been no suggestion that molecules may be incorporated into a polyhedron in locations other than that usually occupied by virus particles.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.